Caspase inhibitors and uses thereof

ABSTRACT

Described herein are compounds that are useful as caspase inhibitors having the formula:  
                 
 
     wherein Ring A is an optionally substituted piperidine, tetrahydroquinoline or tetrahydroisoquinoline ring; R 1  is hydrogen, CN, CHN 2 , R, or CH 2 Y; R is an optionally substituted group selected from an aliphatic group, an aryl group, or an aralkyl group; Y is an electronegative leaving group; R 2  is CO 2 H, CH 2 CO 2 H, or esters, amides or isosteres thereof; and R 3  is hydrogen, an optionally substituted aryl group, an optionally substituted aralkyl group, or an optionally substituted C 1-6  aliphatic group, R 4  is an optionally substituted group selected from an aryl group or a heterocyclyl group, or R 3  and R 4  taken together with the nitrogen to which they are attached optionally form a substituted or unsubstituted monocyclic, bicyclic or tricyclic ring.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional PatentApplication No. 60/232,573, filed Sep. 13, 2000.

FIELD OF THE INVENTION

[0002] This invention is in the field of medicinal chemistry and relatesto novel compounds, and pharmaceutical compositions thereof, thatinhibit caspases that mediate cell apoptosis and inflammation. Theinvention also relates to methods of using the compounds andpharmaceutical compositions of this invention to treat diseases wherecaspase activity is implicated.

BACKGROUND OF THE INVENTION

[0003] Apoptosis, or programmed cell death, is a principal mechanism bywhich organisms eliminate unwanted cells. The deregulation of apoptosis,either excessive apoptosis or the failure to undergo it, has beenimplicated in a number of diseases such as cancer, acute inflammatoryand autoimmune disorders, ischemic diseases and certainneurodegenerative disorders (see generally Science, 1998, 281,1283-1312; Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).

[0004] Caspases are a family of cysteine protease enzymes that are keymediators in the signaling pathways for apoptosis and cell disassembly(Thornberry, Chem. Biol., 1998, 5, R97-R103). These signaling pathwaysvary depending on cell type and stimulus, but all apoptosis pathwaysappear to converge at a common effector pathway leading to proteolysisof key proteins. Caspases are involved in both the effector phase of thesignaling pathway and further upstream at its initiation. The upstreamcaspases involved in initiation events become activated and in turnactivate other caspases that are involved in the later phases ofapoptosis.

[0005] Caspase-1, the first identified caspase, is also known asinterleukin converting enzyme or “ICE.” Caspase-1 converts precursorinterleukin-1β (“pIL-1β”) to the pro-inflammatory active form byspecific cleavage of pIL-1β between Asp-116 and Ala-117. Besidescaspase-1 there are also eleven other known human caspases, all of whichcleave specifically at aspartyl residues. They are also observed to havestringent requirements for at least four amino acid residues on theN-terminal side of the cleavage site.

[0006] The caspases have been classified into three groups depending onthe amino acid sequence that is preferred or primarily recognized. Thegroup of caspases, which includes caspases 1, 4, and 5, has been shownto prefer hydrophobic aromatic amino acids at position 4 on theN-terminal side of the cleavage site. Another group which includescaspases 2, 3 and 7, recognize aspartyl residues at both positions 1 and4 on the N-terminal side of the cleavage site, and preferably a sequenceof Asp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10,tolerate many amino acids in the primary recognition sequence, but seemto prefer residues with branched, aliphatic side chains such as valineand leucine at position 4.

[0007] The caspases have also been grouped according to their perceivedfunction. The first subfamily consists of caspases-1 (ICE), 4, and 5.These caspases have been shown to be involved in pro-inflammatorycytokine processing and therefore play an important role ininflammation. Caspase-1, the most studied enzyme of this class,activates the IL-1β precursor by proteolytic cleavage. This enzymetherefore plays a key role in the inflammatory response. Caspase-1 isalso involved in the processing of interferon gamma inducing factor(IGIF or IL-18) which stimulates the production of interferon gamma, akey immunoregulator that modulates antigen presentation, T-cellactivation and cell adhesion.

[0008] The remaining caspases make up the second and third subfamilies.These enzymes are of central importance in the intracellular signalingpathways leading to apoptosis. One subfamily consists of the enzymesinvolved in initiating events in the apoptotic pathway, includingtransduction of signals from the plasma membrane. Members of thissubfamily include caspases-2, 8, 9 and 10. The other subfamily,consisting of the effector capsases 3, 6 and 7, are involved in thefinal downstream cleavage events that result in the systematic breakdownand death of the cell by apoptosis. Caspases involved in the upstreamsignal transduction activate the downstream caspases, which then disableDNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton andfinally fragment the cell.

[0009] Knowledge of the four amino acid sequence primarily recognized bythe caspases has been used to design caspase inhibitors. Reversibletetrapeptide inhibitors have been prepared having the structureCH₃CO-[P4]-[P3]-[P2]-CH(R)CH₂CO₂H where P2 to P4 represent an optimalamino acid recognition sequence and R is an aldehyde, nitrile or ketonecapable of binding to the caspase cysteine sulfhydryl. Rano andThornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med.Chem. Lett. 3, 2689-2692 (1993); Nicholson et al., Nature 376, 37-43(1995). Irreversible inhibitors based on the analogous tetrapeptiderecognition sequence have been prepared where R is anacyloxymethylketone —COCH₂OCOR′. R′ is exemplified by an optionallysubstituted phenyl such as 2,6-dichlorobenzoyloxy and where R is COCH₂Xwhere X is a leaving group such as F or Cl. Thornberry et al.,Biochemistry 33, 3934 (1994); Dolle et al., J Med. Chem. 37, 563-564(1994).

[0010] The utility of caspase inhibitors to treat a variety of mammaliandiseases associated with an increase in cellular apoptosis has beendemonstrated using peptidic caspase inhibitors. For example, in rodentmodels, caspase inhibitors have been shown to reduce infarct size andinhibit cardiomyocyte apoptosis after myocardial infarction, to reducelesion volume and neurological deficit resulting from stroke, to reducepost-traumatic apoptosis and neurological deficit in traumatic braininjury, to be effective in treating fulminant liver destruction, and toimprove survival after endotoxic shock. Yaoita et al., Circulation, 97,276 (1998); Endres et al., J Cerebral Blood Flow and Metabolism, 18,238, (1998); Cheng et al., J. Clin. Invest., 101, 1992 (1998); Yakovlevet al., J Neuroscience, 17, 7415 (1997); Rodriquez et al., J. Exp. Med.,184, 2067 (1996); Grobmyer et al., Mol. Med., 5, 585 (1999).

[0011] In general, the peptidic inhibitors described above are verypotent against some of the caspase enzymes. However, this potency hasnot always been reflected in cellular models of apoptosis. In additionpeptide inhibitors are typically characterized by undesirablepharmacological properties such as poor oral absorption, poor stabilityand rapid metabolism. Plattner and Norbeck, in Drug DiscoveryTechnologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England,1990).

[0012] Recognizing the need to improve the pharmacological properties ofthe peptidic caspase inhibitors, peptidomimetic and non-natural aminoacid peptide inhibitors have been reported.

[0013] WO 96/40647 discloses ICE inhibitors of the formula:

[0014] wherein B is H or an N-terminal blocking group; R₁ is the aminoacid side chain of the P₁ amino acid residue wherein the P₁ amino acidis not Asp; P_(n) is an amino acid residue or a heterocyclic replacementof the amino acid; R₄ is hydroxyl, alkoxyl, acyl, hydrogen, alkyl orphenyl; m is 0 or a positive integer; and X is N, S, O, or CH₂.

[0015] U.S. Pat. No. 5,585,357 discloses compounds which inhibitinterleukin-1β protease. These inhibitors are represented by theformula:

[0016] wherein each AA is independently L-valine or L-alanine; n is 0-2;R₁ is certain groups; and R₈, R₉, R₁₀ are each independently hydrogen,lower alkyl, halo substituted methyl, carbalkoxy, benzyl, phenyl orphenyl mono- or disubstituted with fluoro, nitro, methoxy, chloro,trifluoromethyl or methanesulfonyl.

[0017] WO 98/16502 discloses aspartate ester inhibitors ofinterleukin-1β converting enzyme of the formula:

[0018] wherein R¹ is, inter alia, R⁵N(R^(a))CHR⁶CO—; R² is certaingroups; R⁶ is H, C₁₋₆ alkyl, —(CH₂)hd n, aryl, —(CH₂)_(n)CO₂R^(a),hydroxyl substituted C₁₋₆ alkyl, or imidazole substituted C₁₋₆ alkyl;and R^(a) is independently hydrogen, C₁₋₆ alkyl or —(CH₂)_(n)aryl

[0019] WO 99/18781 discloses dipeptide apoptosis inhibitors having theformula:

[0020] where R₁ is an N-terminal protecting group; AA is a residue ofany natural α-amino acid, or β-amino acid; and R₂ and R₃ are defined inthe application.

[0021] WO 00/023421 discloses (substituted)acyl dipeptide apoptosisinhibitors having the formula:

[0022] wherein n is 0, 1, or 2; q is 1 or 2; A is a residue of anynatural or non-natural amino acid; B is a hydrogen atom, a deuteriumatom, C1-10 straight chain or branched alkyl, cycloalkyl, phenyl,substituted phentyl, naphthyl, substituted naphthyl, 2-benzoxazolyl,substituted 2-oxazolyl, (CH₂)_(m)cycloalkyl, (CH2)_(m)phenyl,(CH₂)_(m)(substituted phenyl), (CH2)_(m)(1- or 2-naphthyl),(CH₂)_(m)heteroaryl, halomethyl, CO₂R₁₃, CONR₁₄R₁₅, CH₂ZR₁₆, CH₂OCOaryl,CH₂OCO(substituted aryl), CH₂OCO(heteroaryl), CH₂OCO(substitutedheteroaryl), or CH₂OPO(R₁₇)R₁₈, where R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ and R₁₈are defined in the application; R₂ is selected from a group containinghydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, (CH₂)_(m)NH₂;R₃ is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, orsubstituted phenylalkyl; X is CH₂, C═O, O, S, NH, C═ONH or CH₂OCONH; andZ is an oxygen or a sulfur atom.

[0023] WO 00/061542 discloses dipeptide apoptosis inhibitors having theformula:

[0024] where R₁ is an optionally substituted alkyl or hydrogen group; R₂is hydrogen or optionally substituted alkyl; Y is a residue of a naturalor non-natural amino acid and R₃ is an alkyl, saturated carbocyclic,partially saturated carbocyclic, aryl, saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted; X is O, S, NR₄, or (CR₄R₅)_(n) where R₄ and R₅are, at each occurrence, independently selected from the groupconsisting of hydrogen, alkyl and cycloalkyl, and n is 0, 1, 2, or 3; orX is NR₄, and R₃ and R₄ are taken together with the nitrogen atom towhich they are attached to form a saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted or X is CR₄R₅, and R₃ and R₄ are taken togetherwith the carbon atom to which they are attached to form a saturatedcarbocyclic, partially saturated carbocyclic, aryl, saturatedheterocyclic, partially saturated heterocyclic or oxygen-containingheteroaryl group, wherein said group is optionally substituted; andprovided that when X is O, then R₃ is not unsubstituted benzyl ort-butyl; and when X is CH₂, then R₃ is not H.

[0025] While a number of caspase inhibitors have been reported, it isnot clear whether they possess the appropriate pharmacologicalproperties to be therapeutically useful. Therefore, there is a continuedneed for small molecule caspase inhibitors that are potent, stable, andpenetrate membranes to provide effective inhibition of apoptosis invivo. Such compounds would be extremely useful in treating theaforementioned diseases where caspase enzymes play a role.

SUMMARY OF THE INVENTIONS

[0026] It has now been found that compounds of this invention andpharmaceutical compositions thereof are particularly effective asinhibitors of caspases and cellular apoptosis. These compounds have thegeneral formula I:

[0027] wherein:

[0028] Ring A is an optionally substituted piperidine,tetrahydroquinoline or tetrahydroisoquinoline ring;

[0029] R¹ is hydrogen, CN, CHN₂, R, or CH₂Y;

[0030] R is an optionally substituted group selected from an aliphaticgroup, an aryl group, or an aralkyl group;

[0031] Y is an electronegative leaving group;

[0032] R² is CO₂H, CH₂CO₂H, or esters, amides or isosteres thereof; and

[0033] R³ is hydrogen, an optionally substituted aryl group, anoptionally substituted aralkyl group, or an optionally substituted C₁₋₆aliphatic group, R ⁴ is an optionally substituted group selected from anaryl group or a heterocyclyl group, or R³ and R⁴ taken together with thenitrogen to which they are attached optionally form is a substituted orunsubstituted monocyclic, bicyclic or, tricyclic ring.

[0034] The compounds of this invention have potent inhibition propertiesacross a range of caspase targets with good efficacy in cellular modelsof apoptosis. In addition, these compounds are expected to have improvedcell penetration and pharmacokinetic properties and, as a consequence oftheir potency, have improved efficacy against diseases where caspasesare implicated.

DETAILED DESCRIPTION OF THE INVENTION

[0035] This invention provides novel compounds, and pharmaceuticallyacceptable derivatives thereof, that are particularly effective ascaspase inhibitors. The invention also provides methods for using thecompounds to treat caspase-mediated diseases in mammals. The compoundshave the general formula I:

[0036] wherein:

[0037] Ring A is an optionally substituted piperidine,tetrahydroquinoline or tetrahydroisoquinoline ring;

[0038] R¹ is hydrogen, CN, CHN₂, R, or CH₂Y;

[0039] R is an optionally substituted group selected from an aliphaticgroup, an aryl group, or an aralkyl group;

[0040] Y is an electronegative leaving group;

[0041] R² is CO₂H, CH₂CO₂H, or esters, amides or isosteres thereof; and

[0042] R³ is hydrogen, an optionally substituted aryl group, anoptionally substituted aralkyl group, or an optionally substituted C₁₋₆aliphatic group, R⁴ is an optionally substituted group selected from anaryl group or a heterocyclyl group, or R³ and R⁴ taken together with thenitrogen to which they are attached optionally form a substituted orunsubstituted monocyclic, bicyclic or tricyclic ring.

[0043] As used herein, the following definitions shall apply unlessotherwise indicated. The phrase “optionally substituted” may be usedinterchangeably with the phrase “substituted or unsubstituted” or withthe term “(un)substituted.” Unless otherwise indicated, an optionallysubstituted group may have one or more substituents that areindependently selected.

[0044] The term “aliphatic” as used herein means straight chained,branched or cyclic C₁-C₁₂ hydrocarbons which are completely saturated orwhich contain one or more units of unsaturation. For example, suitablealiphatic groups include substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl, or alkynyl groups and hybrids thereof such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. The term“alkyl” used alone or as part of a group or larger moiety refers to bothstraight and branched chains containing one to twelve carbon atoms.

[0045] The term “halogen” means F, Cl, Br, or I. The term “heteroatom”means nitrogen, oxygen or sulfur.

[0046] The term “aryl” refers to monocyclic or polycyclic aromaticgroups, and monocyclic or polycyclic heteroaromatic groups containingone or more heteroatoms, having five to fourteen atoms. Such groupsinclude, but are not limited to, phenyl, naphthyl, anthryl, furanyl,thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl,pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl,indolyl, isoindolyl, indolinyl, benzofuranyl, benzothiophenyl,indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolizinyl,quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydrofuranyl,phthalimidinyl, tetrazolyl, and chromanyl.

[0047] The term “heterocyclic group” refers to saturated and partiallyunsaturated monocyclic or polycyclic ring systems containing one or moreheteroatoms, wherein a monocyclic ring preferably has 5-7 ring atoms anda polycyclic ring preferably has 8-14 ring atoms. Such groups include,but are not limited to aziranyl, oxiranyl, azetidinyl,tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, dioxolanyl, imidazolinyl,imidazolidinyl, pyrazolinyl, pyrazolidinyl, pyranyl, piperidinyl,dioxanyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl,trithianyl, quinuclidinyl, oxepanyl, and thiepanyl. The term“heterocyclic ring”, whether saturated or unsaturated, also refers torings that are optionally substituted.

[0048] An aryl group (including heteroaryl groups) or an aralkyl group,such as benzyl or phenethyl, may contain one or more substituents.Examples of suitable substituents on the unsaturated carbon atom of anaryl group include halogen, —R^(o), —OR^(o), —SR^(o),1,2-methylene-dioxy, 1,2-ethylenedioxy, protected OH (such as acyloxy),phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph), —CH₂(Ph),substituted —CH₂(Ph), —CH₂CH₂(Ph), substituted —CH₂CH₂(Ph), —NO₂, —CN,—N(R^(o))₂, —NR^(o)C(O)R^(o), —NR^(o)C(O)N(R^(o))₂, —NR^(o)CO₂R^(o),—NR^(o)NR^(o)C(O)R^(o), —NR^(o)NR^(o)C(O)N(R^(o))₂,—NR^(o)NR^(o)CO₂R^(o), —C(O)C(O)R^(o), —C(O)CH₂C(O)R^(o), —CO₂R^(o),—C(O)R^(o), —C(O)N(R^(o))₂, —OC(O)N(R^(o))₂, —S(O)₂R^(o), —SO₂N(R^(o))₂,—S(O)R^(o), —NR^(o)SO₂N(R^(o))₂, —NR^(o)SO₂R^(o), —C(═S)N(R^(o))₂,—C(═NH)—N(R^(o))₂, —(CH₂)_(y)NHC(O)R^(o),—(CH₂)_(y)NHC(O)CH(V-R^(o))(R^(o)); wherein R^(o) is H, a substituted orunsubstituted aliphatic group, preferably having 1-3 carbons, anunsubstituted heteroaryl or heterocyclic ring, phenyl (Ph), substitutedPh, —O(Ph), substituted —O(Ph), —CH₂(Ph), or substituted —CH₂(Ph); y is0-6; and V is a linker group. Examples of substituents on the aliphaticgroup or the phenyl ring include amino, alkylamino, dialkylamino,aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano,carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, orhaloalkyl.

[0049] An aliphatic group or a non-aromatic heterocyclic ring maycontain one or more substituents. Examples of suitable substituents onthe saturated carbon of an aliphatic group or of a non-aromaticheterocyclic ring include those listed above for the unsaturated carbonas well as the following: ═O, ═S, ═NNHR*, ═NN(R*)₂, ═N—, ═NNHC(O)R*,═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR*, where each R* is independentlyselected from hydrogen, an unsubstituted aliphatic group or asubstituted aliphatic group. Examples of substituents on the aliphaticgroup include amino, alkylamino, dialkylamino, aminocarbonyl, halogen,alkyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy,dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl,alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl.

[0050] A substitutable nitrogen on an aromatic or non-aromaticheterocyclic ring may be optionally substituted. Suitable substituentson the nitrogen include —R⁺, —N(R⁺)₂, —C(O)R⁺, —CO₂R⁺, —C(O)C(O)R⁺,—C(O)CH₂C(O)R⁺, —SO₂R⁺, —SO₂N(R⁺)₂, —C(═S)N(R⁺)₂, —C(═NH)—N(R⁺)₂, and—NR⁺SO₂R⁺; wherein R⁺ is H, an aliphatic group, a substituted aliphaticgroup, phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph), CH₂(Ph),substituted CH₂(Ph), or an unsubstituted heteroaryl or heterocyclicring. Examples of substituents on the aliphatic group or the phenyl ringinclude amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy,dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl,alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl. Nitrogen and sulfurmay be in their oxidized form, and nitrogen may be in a quaternizedform.

[0051] The term “electronegative leaving group” has the definition knownto those skilled in the art (see March, Advanced Organic Chemistry,4^(th) Edition, John Wiley & Sons, 1992). Examples of electronegativeleaving groups include halogens such as F, Cl, Br, I, aryl- andalkylsulfonyloxy groups, trifluoromethanesulfonyloxy, —SR, —OPO(R⁵)(R⁶), where R is an aliphatic group, an aryl group, an aralkyl group, acarbocyclyl group, a carbocyclylalkyl group, a heterocyclyl group, or aheterocyclylalkyl group, and R⁵ and R⁶ are independently selected from Ror OR.

[0052] When the R² group is in the form of an ester or amide, thepresent compounds undergo metabolic cleavage to the correspondingcarboxylic acids, which are the active caspase inhibitors. Because theyundergo metabolic cleavage, the precise nature of the ester or amidegroup is not critical to the working of this invention. The structure ofthe R² group may range from the relatively simple diethyl amide to asteroidal ester. Examples of the ester alcohol moiety of R² carboxylicacids include, but are not limited to, alcohols of C₁₋₁₂aliphaticgroups, such as C₁₋₆ alkyl or C₃₋₁₀ cycloalkyl, aryl groups, such asphenyl, aralkyl groups, such as benzyl or phenethyl, heterocyclyl orheterocyclylalkyl groups. Examples of suitable R² heterocyclyl groupsinclude, but are not limited to, 5-6 membered heterocyclic rings havingone or two heteroatoms such as piperidinyl, piperazinyl, or morpholinyl.

[0053] Amides of R² carboxylic acids may be primary, secondary ortertiary. Suitable substituents on the amide nitrogen include, but arenot limited to, one or more groups independently selected from thealiphatic, aryl, aralkyl, heterocyclyl or heterocyclylalkyl groupsdescribed above for the R² ester alcohol. Likewise, other prodrugs areincluded within the scope of this invention. See Bradley D. Anderson,“Prodrugs for Improved CNS Delivery” in Advanced Drug Delivery Reviews(1996), 19, 171-202.

[0054] Isosteres or bioisosteres of carboxylic acids and esters oramides result from the exchange of an atom or group of atoms to create anew compound with similar biological properties to the parent carboxylicacid or ester. The bioisosteric replacement may be physicochemically ortopologically based. An example of an isosteric replacement for acarboxylic acid is CONHSO₂(alkyl) such as CONHSO₂Me.

[0055] Compounds of this invention where R² is CO₂H or CH₂CO₂H,γ-ketoacids or δ-ketoacids respectively, may exist in solution as eitherthe open form (a) or the cyclized hemiketal form (b) (y=1 forγ-ketoacids, y=2 for δ-ketoacids). The representation herein of eitherisomeric form is meant to include the other.

[0056] Likewise it will be apparent to one skilled in the art thatcertain compounds of this invention may exist in tautomeric forms orhydrated forms, all such forms of the compounds being within the scopeof the invention. Unless otherwise stated, structures depicted hereinare also meant to include all stereochemical forms of the structure;i.e., the R and S configurations for each asymmetric center. Therefore,single stereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

[0057] One embodiment of this invention relates to compounds of formulaI wherein Ring A is an optionally substituted piperidine ring,represented by formula Ia:

[0058] Another embodiment of this invention relates to compounds offormula I wherein Ring A is an optionally substitutedtetrahydroquinoline ring, represented by formula Ib below:

[0059] Another embodiment of this invention relates to compounds offormula I wherein Ring A is an optionally substitutedtetrahydroisoquinoline ring are represented by formula Ic below:

[0060] Ring A may be substituted or unsubstituted. Examples of suitableRing A substituents include one or more groups selected from halogen,—R, —OR, —OH, —SR, protected OH (such as acyloxy), phenyl (Ph),substituted Ph, —OPh, substituted —OPh, —NO₂, —CN, —NH₂, —NHR, —N(R)₂,—NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R, —CO₂H, —COR,—CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR, —NHS(O)₂R, ═O, ═S,═NNHR, ═NNR₂, ═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR where R is analiphatic group or a substituted aliphatic group. Preferably R is a C₁₋₆aliphatic group.

[0061] A preferred R¹ group is CH₂Y where Y is an electronegativeleaving group. Most preferably Y is F.

[0062] R³ and R⁴ may be taken together with the nitrogen to which theyare attached to form a substituted or unsubstituted monocyclic, bicyclicor tricyclic ring. The R³R⁴N ring system may be aromatic or non-aromaticand will have 1-6 heteroatoms selected from oxygen, nitrogen or sulfur.Preferably each ring of the R³R⁴N ring system has 5-7 ring atomsExamples of such rings include indole, isoindole, indoline, indazole,purine, benzimidazole, benzthiazole, imidazole, imidazoline, thiazole,pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine,triazole, piperidine, morpholine, thiomorpholine, piperazine, carbazole,phenothiazine, phenoxazine, phenanthridine, acridine, purine,quinolizine, quinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, quinuclidine,and phenazine.

[0063] Preferred compounds of this invention have one or more, and morepreferably all, of the features selected from the group consisting of:

[0064] (a) R¹ is CH₂Y wherein Y is an electronegative leaving group;

[0065] (b) R² is CO₂H or an ester or isosteres thereof; and

[0066] (c) R³ is hydrogen, an optionally substituted aryl group, anoptionally substituted aralkyl group or an optionally substituted C₁₋₆aliphatic group, R⁴ is an optionally substituted group selected from anaryl group a heterocyclyl group, or R³ and R⁴, taken together with thenitrogen to which they are attached, optionally form a ring selectedfrom the group consisting of indole, isoindole, indoline, indazole,purine, benzimidazole, benzthiazole, imidazole, imidazoline, thiazole,pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine,triazole, piperidine, morpholine, thiomorpholine, piperazine, carbazole,phenothiazine, phenoxazine, phenanthridine, acridine, purine,quinolizine, quinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, quinuclidine,and phenazine. A preferred R³ or R⁴ aryl group is phenyl or a 5-6membered heteroaromatic ring containing 1-3 heteroatoms selected fromnitrogen, oxygen or sulfur. A preferred R³ aralkyl group is a C₁₋₃alkylidene chain substituted with a preferred aryl group. Preferred R⁴heterocyclyl groups include 5-6 membered rings containing 1-3heteroatoms selected from nitrogen, oxygen or sulfur.

[0067] More preferred are compounds where Y is F and R³ and R⁴, takentogether with the nitrogen to which they are attached, optionally form aring selected from the group consisting of indole, isoindole, indoline,indazole, purine, benzimidazole, benzthiazole, imidazole, imidazoline,thiazole, pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline,pyrazolidine, triazole, piperidine, morpholine, thiomorpholine,piperazine, carbazole, phenothiazine, phenoxazine, phenanthridine,acridine. purine, quinolizine, quinoline, isoquinoline, cinnoline,phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, pteridine,quinuclidine, and phenazine. Even more preferred are compounds whereinR³R⁴N— ring system is carbazole, piperidine, indole, dihydroindole,phenothiazine, dihydrophenanthridine, phenoxazine, acridine,acridin-9-one, β-carboline, or 9-thia-2,10-diaza-anthracene. Mostpreferred are compounds wherein R³R⁴N— ring system is carbazole,phenothiazine, or dihydrophenanthridine.

[0068] The R³R⁴N— ring system may be optionally substituted. Examples ofsuitable substituents on the R³R⁴N— ring include one or more groupsselected from halogen, —R, —OR, —OH, —SR, protected OH (such asacyloxy), phenyl (Ph), substituted Ph, —OPh, substituted —OPh, —NO₂,—CN, —NH₂, —NHR, —N(R)₂, —NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R,—CO₂R, —CO₂H, —COR, —CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR,—NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂, ═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or═NR where R is an aliphatic group or a substituted aliphatic group.Preferably R is a C₁₋₆ aliphatic group.

[0069] Representative examples of compounds of the present invention areshown below in Table 1. TABLE 1 Representative Compounds Ia

Ib

Ic

Ring A No. R¹ R² Type R³R⁴N- I-1 CH₂F CO₂H Ia

I-2 CH₂F CO₂H Ia

I-3 CH₂F CO₂H Ia

I-4 CH₂F CO₂H Ia

I-5 CH₂F CO₂H Ia

I-6 CH₂F CO₂H Ia

I-7 CH₂F CO₂H Ia

I-8 CH₂F CO₂H Ia

I-9 CH₂F CO₂H Ia

I-10 CH₂F CO₂H Ia

I-11 CH₂F CO₂NH₂ Ia

I-12 CH₂F CO₂NHEt Ia

I-13 CH₂F CO₂NEt₂ Ia

I-14 CH₂F CONHCH₂CH₂N(CH₃)₂ Ia

I-15 CH₂F CO₂H Ib

I-16 CH₂F CO₂H Ic

I-17 CH₂F CO₂H Ib

I-18 CH₂F CO₂H Ic

I-19 CH₂F CO₂H Ia

I-20 CH₂F CO₂H Ia

I-21 CH₂F CO₂H Ia

I-22 CH₂F CO₂H Ia

I-23 CH₂F CO₂H Ia

I-24 CH₂F CO₂H Ia

I-25 CH₂F CO₂H Ia

I-26 CH₂F CO₂H Ia

I-27 CH₂F CO₂H Ia

I-28 CH₂F CO₂H Ia

I-29 CH₂F CO₂H Ia

I-30 CH₂F CO₂H Ia

[0070] The compounds of this invention may be prepared in general bymethods known to those skilled in the art for analogous compounds, asillustrated by the general Scheme I below and by the preparativeexamples that follow.

[0071] Reagents: (a) H- (Ring A) -CO₂R; (b) NaOH, THF, H₂O; (c)H₂NCH(CH₂R²)CH(OH)R¹; EDC, N,N-dimethylaminopyridine,1-hydroxybenzotriazole; (d) i. Dess-Martin periodinane; ii.trifluoroacetic acid, dichloromethane.

[0072] Scheme I above shows a general route for preparing compounds ofthis invention. The carbamoyl chloride 1 (or an analogous isocyanate)may be coupled with an amino acid ester, H- (Ring A) -CO₂R to provideurea 2. Hydrolysis of the ester 2 provides acid 3. If the ester is atert-butyl ester then it may be hydrolyzed with acid such astrifluoroacetic acid. The acid 3 may then be coupled with an appropriateamino alcohol, H₂NCH(CH₂R²)CH(OH)R¹ to provide 4. In step c, “EDC” is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Depending on the natureof R¹ an amino ketone may be used, in place of the amino alcohol, whichavoids the subsequent oxidation step. In the case of fluoromethylketones where R² is CO₂tBu, the amino alcohol may be obtained accordingto the method of Revesz et al., Tetrahedron Lett., 1994, 35, 9693. Thehydroxyl in compound 4 is oxidized to compound I which may be furthermodified, depending on the nature of R², according to methods generallyknown in the art for analogous compounds. For example, if the product Irequires R² to be a carboxylic acid, then R² in 4 is preferably an esterand the further step is a hydrolysis of the ester group.

[0073] Certain useful intermediates for making compounds of thisinvention may be obtained as follows. Substituted phenothiazines areeither commercially available or may be prepared as described by J. I.G. Cadogan, S. Kulik, C. Thomson and M. J. Todd, J. Chem. Soc., 1970,2437-2441. 9,10-Dihydrophenanthridine was prepared according to G. M.Badger, J. H. Seidler and B. Thomson, J. Chem. Soc, 1951, 3207-3211.Carbamoyl chlorides are either commercially available or may be preparedas described by R. Dahlbom and B. Bjorkqvist, Acta Chem. Scand., 15,1961, 2043-2046.

SYNTHETIC EXAMPLES Example 1 [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid

[0074]

[0075] Method A

(S)-(1-Phenothiazine-10-carbonyl)piperidine-2-carboxylic acid methylester

[0076]

[0077] To a stirred solution of methyl pipecolate hydrochloride (1 g,5.57 mmol) in THF (10 ml) was added phenothiazine carbonyl chloride(1.457 g, 5.57 mmol) followed by diisopropylethylamine (2.02 mL, 11.68mmol). The resulting solution was stirred for 16 h before beingpartitioned between ethyl acetate and aq. sat. NH₄Cl. The organic layerwas washed with brine, dried (MgSO₄), filtered and evaporated. Theresidue was purified by flash chromatography (15% ethyl acetate inhexane) to afford the sub-title compound as a colorless oil whichcrystallized upon standing (1.823 g, 89%): ¹H NMR (400 MHz, CDCl₃)δ1.13-1.48 (3H, m), 2.57-2.69 (2H, m), 2.16 (1H, m), 3.00 (1H, m), 3.74(4H, s+m), 5.00 (1H, m), 7.11 (2H, t), 7.22-7.34 (4H, m), 7.76 (2H, d);¹³C NMR (100 MHz, CDCl₃) δ21.3 (CH₂), 24.8 (CH₂), 27.3 (CH₂), 44.9(CH₂), 52.5 (CH₃), 55.9 (CH), 122.8 (CH), 125.5 (CH), 127.8 (CH), 128.0(CH), 129.2 (C), 141.7 (C), 158.4 (C), 172.2 (C).

[0078] Method B

(S)-(1-Phenothiazine-10-carbonyl)piperidine-2-carboxylic acid

[0079]

[0080] To a stirred solution of (S)-(1-phenothiazine-10-carbonyl)piperidine-2-carboxylic acid methyl ester (0.912 g) in THF(15 ml) and water (8 ml) was added 2M NaOH (3.71 mL) and the reactionmixture was stirred for 16 hours. The reaction mixture was poured intosodium hydrogen carbonate solution (50 ml) and extracted with ethylacetate (40 ml). Aqueous phase made acidic and extracted with ethylacetate (2×75 ml). Organic extracts combined, dried (MgSO₄) andconcentrated to eave the subtitle compound as a white solid (0.709 g,81%): ¹H NMR (400 MHz, CDCl₃) δ0.99-1.72 (5H, m), 2.23 (1H, m), 2.97(1H, m), 3.58 (1H, m), 4.93 (1H, m), 7.16 (2H, t), 7.28 (2H, t), 7.37(2H, d), 7.78 (2H, d); ¹³C NMR (100 MHz, CDCl₃) δ21.0 (CH₂), 24.2 (CH₂),26.7 (CH₂), 45.7 (CH₂), 56.0 (CH), 123.8 (CH), 126.0 (CH), 127.9 (CH),128.1 (CH), 130.3 (C), 141.2 (C), 160.1 (C), 175.9 (C).

[0081] Method C

[3S/R, 4S/R(2S)]-5-Fluoro-4-hydroxy-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid tertbutyl ester

[0082]

[0083] A stirred mixture of(S)-(1-phenothiazine-10-carbonyl)piperidine-2-carboxylic acid (233 mg,0.658 mmol), 3-amino-5-fluoro-4-hydroxy-pentanoic acid tert-butyl ester(150 mg, 0.724 mmol), HOBt (98 mg, 0.724 mmol), DMAP (88 mg, 0.724 mmol)and anhydrous THF (10 mL) was cooled to 0° C. then EDC (139 mg, 0.724mmol) was added. The mixture was allowed to warm to room temperatureduring 16 h then concentrated under reduced pressure. The residue waspurified by flash chromatography (50% ethyl acetate in hexane) to affordthe sub-title compound as a pale pink foam (294 mg, 82%): ¹H NMR (400MHz, CDCl₃) δ1.96 (1H, m), 1.18-1.60 (13H, m), 2.10-2.25 (1H, m),2.48-2.70 (2H, m), 2.78-2.94 (1H, m), 3.51-4.72 (7H, m), 7.03-7.36 (7H,m), 7.71-7.76 (2H, m); ¹⁹F (376 MHz, CDCl₃) δ−228.9 (t), −229.3 (t),−230.1 (t), −230.2 (t).

[0084] Method D

[3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid tert-butyl ester

[0085]

[0086] A stirred solution of [3S/R, 4S/R(2S)]-5-fluoro-4-hydroxy-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid tertbutyl ester (294 mg, 0.541 mmol) in anhydrous DCM (10 mL) wastreated with 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (344mg, 0.812 mmol) at 0° C. The resulting mixture was allowed to warm toroom temperature over 2 h, diluted with ethyl acetate, then poured intoa 1:1 mixture of saturated aqueous sodium hydrogen carbonate andsaturated aqueous sodium thiosulphate. The organic layer was removed andthe aqueous layer re-extracted with ethyl acetate. The combined organicextracts were dried (Na₂SO₄) and concentrated. The residue was purifiedby flash chromatography (30% ethyl acetate in hexane) to afford thesub-title compound as a pale pink foam (220 mg, 75%): ¹H NMR (400 MHz,CDCl₃) δ0.84-0.96 (1H, m), 1.20-1.40 (10H, m+2s), 1.51-1.56 (3H, m),2.20-2.27 (1H, m), 2.70-2.98 (3H, m), 3.49-3.63 (1H, m), 4.74-5.24 (4H,m), 7.14-7.18 (2H, m), 7.28-7.38 (4H, m), 7.48-7.79 (3H, m); ¹³C (100MHz, CDCl₃) δ20.8/21.0 (CH₂), 23.7/23.9 (CH₂), 25.8/25.9 (CH₂),28.2/28.3 (CH₃), 36.8/36.9 (CH₂), 46.0/46.1 (CH₂), 52.9 (CH), 56.8 (CH),82.6 (C), 84.4/84.5 (2d, J 184.0/183.3, CH₂F), 123.7/123.8 (CH), 126.1(CH), 128.0/128.1 (CH), 128.2/128.3 (CH), 130.4/130.5 (C), 141.4 (C),160.0 (C), 170.0 (C), 171.7 (C), 202.9 (C); ¹⁹F (376 MHz, CDCl₃) δ-231.9(t), −232.2 (t).

[0087] Method E

[3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid

[0088]

[0089] Trifluoroacetic acid (5 mL) was added to a stirred ice coldsolution of [3S/R,(2S)]-5-fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid tert-butyl ester (130 mg, 0.24 mmol) in anhydrous DCM (5 mL). Themixture was stirred at 0° C. for 0.5 h then at room temperature for0.5h. The mixture was concentrated under reduced pressure and then theresidue was dissolved in dry DCM. This process was repeated severaltimes in order to remove excess trifluoroacetic acid. The gum waslyophilized twice from HPLC grade water to afford the title compound asa white powder (77 mg, 66%): IR (solid) 1670, 1716, 1782 cm⁻¹; ¹H NMR(400 MHz, d₆-DMSO) δ0.96-0.99 (1H, m), 1.23-1.26 (2H, m), 1.42-1.44 (1H,m), 1.60 (1H, m), 1.91-1.98 (1H, m), 2.51-2.89 (2H, m), 3.11-3.22 (1H,m), 3.57-3.60 (1H, m), 4.30-4.72 and 5.05-5.29 (4H, 2m), 7.11-7.17 (2H,m), 7.24-7.30 (2H, m), 7.34-7.38 (2H, m), 7.57-7.63 (2H, m), 8.07-8.61(1H, m); ¹³C NMR (100 MHz, DMSO) δ(DMSO+TFA) 18.8-/18.9 (CH₂), 22.2/22.3(CH₂), 25.8/26.1 (CH₂), 31.5/33.2 (CH₂), 43.2 (CH₂), 50.6/51.1 (CH),54.4/54.5 (CH), 82.8/82.9 (2d, J 178.6/178.1, CH₂F), 119.9/120.0 (CH),120.4/120.5 (CH), 124.0/124.1 m(CH), 125.9/126.0 (C), 126.4/126.5 (CH),139.6/139.7 (C), 156.0/156.4 (CO), 170.3 (CO), 170.7/170.8 (CO),202.2/202.3 (2d, J 14.6/15.1, CO).; ¹⁹F (376 MHz, DMSO) δ chemical shift(multiplicity, relative intensity) −226.7 (t, 3), −226.9 (t, 3), −230.4(t, 1), −231.2 (t, 1), −232.7 (t, 10), −233.0 (t, 10).

Example 2 [3S/R, (2S)]-3-{[1-(2-Chlorophenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoic acid

[0090]

[0091] This was prepared from 2-chlorophenothiazine carbonyl chlorideusing procedures similar to those described above in Methods A-E (73 mg,69%): IR (solid, cm−1) 1738, 1660, 1555, 1363, 1224 ; ¹H NMR (400 MHz,d₆-DMSO+TFA) δ0.98-1.61 (4H, m), 1.94-2.03 (1H, m), 2.53-2.89 (2H, m),3.12-3.24 (1H, m), 3.51-3.61 (1H, m), 4.31-4.73 and 5.10-5.24 (4H, 2m),7.15-7.49 (6H, m), 7.77-7.81 (1H, m), 8.13-8.64 (1H, m) ; ¹³C NMR (100MHz, DMSO+TFA) δ18.7/18.8 (CH₂), 22.3/22.6 (CH₂), 25.9/26.2 (CH₂),31.5/33.2 (CH₂), 43.0/43.2 (CH₂), 50.6/51.1 (CH), 54.4/54.5 (CH),82.8/82.9 (2d, J 178.7/178.3, CH₂F), 119.3/119.8 (CH), 120.2/120.3 (CH),123.6/123.7 (CH), 124.4/124.5 (CH), 124.6/124.8 (C), 126.6 (CH), 126.9(CH), 127.5 (CH), 131.0 (C), 139.2/139.2 (C), 140.7/140.7 (C),155.5/155.9 (C), 170.1/170.2 (C), 170.7/170.8 (C), 201.2/201.3 (2d, J14.3/13.9, CO) ; ¹⁹F NMR (376 MHz, DMSO+TFA) δ−226.7 (t), −226.9 (t),−230.3 (t), −232.7 (t), −233.0 (t)

EXAMPLE 3 [3S/R,(2S)]-3-{[1-(3-Chlorophenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoicacid

[0092]

[0093] This was prepared from 3-chlorophenothiazine carbonyl chlorideusing procedures similar to those described above in Methods A-E (108mg, 65%): IR (solid, cm−1) 1737, 1655, 1455, 1373, 1224 ; ¹H NMR (400MHz, d₆-DMSO+TFA) δ0.99-1.61 (5H, m), 1.91-2.04 (1H, m), 2.54-2.90 (2H,m), 3.12-3.24(1H, m), 3.48-3.60 (1H, m), 4.26-5.28 (4H, m), 7.15-7.68(7H, m), 8.10-8.62 (1H, m) ; ¹³C NMR (100 MHz, DMSO+TFA) δ18.8 (CH₂),22.2/22.3 (CH₂), 25.8 (CH₂), 33.1/33.2 (CH₂), 43.2 (CH₂), 50.6/51.0(CH), 54.3/54.4 (CH), 82.7/82.8 (2d, CH₂F), 120.2/120.3 (CH),121.3/121.4 (CH), 124.2/124.3 (CH), 124.8/125.0 (C), 125.7 (CH), 126.3(CH), 126.6 (CH), 126.8 (CH), 127.7/127.9 (C), 127.9/128.0 (C), 138.5(C), 139.3 (C), 156.0 (CO), 170.1 (CO), 170.6/170.7 (CO), 201.1/201.2(2d, CO).; ¹⁹F NMR(376 MHz, DMSO+TFA) δ−226.6 (t), −226.9 (t), −232.6(t), −232.9 (t),

EXAMPLE 4 [3S/R,(2S)]-3-{[1-(3,4-Dichlorophenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoicacid

[0094]

[0095] The title compound was prepared from 3,4-dichlorophenothiazinecarbonyl chloride using procedures similar to those described above inMethods A-E (91 mg, 66%): IR (solid, cm−1) 1737, 1439, 1363, 1219 ; ¹HNMR (400 MHz, d₆-DMSO+TFA) δ1.03-1.62 (5H, m), 1.97-2.06 (1H, m),2.54-2.86 (2H, m), 3.14-3.28 (1H, m), 3.59-3.66 (1H, m), 4.30-5.26 (4H,m), 7.15-7.68 (6H, m), 8.14-8.96 (1H, m) ; ¹³C NMR (100 MHz, DMSO+TFA)δ20.2 (CH₂), 23.8 (CH₂), 27.3 (CH₂), 34.6/34.7 (CH₂), 44.5 (CH₂),52.1/52.5 (CH), 55.7/55.9 (CH), 84.2/84.3 (2d, CH₂F), 120.2/120.3 (CH),120.8/120.9 (CH), 124.2/124.4 (C), 125.9 (CH), 127.7/127.8 (C), 128.2(CH), 128.4/128.5 (C), 128.8 (CH), 128.9 (CH), 140.0 (C), 140.1 (C),140.6 (C), 156.8/156.8 (CO), 171.5 (CO), 172.1/172.1 (CO), 202.6/202.7(2d, CO) ; ¹⁹F NMR(376 MHz, DMSO+TFA) δ−226.6 (t), −226.8 (t), −232.6(t), −232.9 (t).

EXAMPLE 5 [3S/R,(2S)]-3-{[1-(2,6-Dichlorophenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoicacid

[0096]

[0097] The title compound was prepared from 2.7-dichlorophenothiazinecarbonyl chloride using procedures similar to those described above inMethods A-E (91 mg, 70%): IR (solid, cm−1) 1737, 1660, 1555, 1363, 1224;¹H NMR (400 MHz, d₆-DMSO+TFA) δ1.02-1.62 (5H, m), 1.91-2.02 (1H, m),2.53-2.90 (2H, m), 3.13-3.25 (1H, m), 3.51-3.62 (1H, m), 4.31-5.29 (4H,m), 7.22-7.75 (6H, m), 8.18-8.65 (1H, m) ; ¹³C NMR (100 MHz, DMSO+TFA)δ20.2 (CH₂), 23.8 (CH₂), 27.3 (CH₂), 34.6 (CH₂), 44.7 (CH₂), 52.5 (CH),55.8 (CH), 84.3 (d, J 178.2, CH₂F), 120.7/121.2 (CH), 122.7/122.8 (CH),124.7/125.1 (C), 125.3/125.4 (CH), 127.4 (CH), 128.1 (CH), 128.7/128.9(C), 129.1 (CH), 129.8 (C), 132.7 (C), 139.5/139.6 (C), 141.8/141.9 (C),157.0 (CO), 171.5 (CO), 172.1 (CO), 202.6 (d, J 14.3, CO); ¹⁹F NMR(376MHz, DMSO+TFA) δ−226.6 (t), −226.9 (t), −232.6 (t), −232.9 (t).

EXAMPLE 6 [3S/R, (2S)]-3-{[1-(Carbazole-9-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoic acid

[0098]

[0099] The title compound was prepared from 9-carbazole carbonylchloride using procedures similar to those described above in MethodsA-E (180 mg, 75%): IR (solid, cm−1) 1737, 1655, 1419, 1373, 1224 ; ¹HNMR (400 MHz, d₆-DMSO+TFA) δ1.36-1.65 (6H, m), 1.94-1.99 (1H, m),2.12-2.21 (1H, m), 2.59-2.89 (2H, m), 4.32-5.27 (4H, m), 7.30-7.36 (2H,m), 7.48-7.54 (2H, m), 7.63-7.76 (2H, m), 8.17-8.72 (3H, m) ;¹³ C NMR(100 MHz, DMSO+TFA) δ19.0 (CH₂), 23.7/23.8 (CH₂), 26.5/26.8 (CH₂),33.3/33 .5 (CH₂), 44.1 (br, CH₂), 50.9/51.4 (CH), 54.5 (br,CH),82.9/83.1 (2d, J 178.7/178.7, CH₂F), 111.0/111.1 (CH), 111.9 (CH),119.5/119.7 (CH), 120.6/120.7 (CH), 122.5/122.7 (C), 125.8/125.9 (CH),137.1/137.4 (C), 153.2/153.3 (C), 170.3/170.4 (C), 170.8/170.9 (C),201.4/201.5 (2d, J 14.6/14.6, CO) ; ¹⁹F NMR (376 MHz, DMSO+TFA) δd (J,%I) −226.6 (t, 3), −226.8 (t, 3), −230.0 (t, 1), −232.7 (t, 10), −232.7(t, 10).

EXAMPLE 7 [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(6H-phenanthridine-5-carbonyl)-piperidine-2-carbonyl]amino}-pentanoicacid

[0100]

[0101] The title compound was prepared from 9,10-dihydrophenanthrininecarbonyl chloride using procedures similar to those described above inMethods A-E (115 mg, 61%): IR (solid, cm−1) 1731, 1419, 1363, 1219; ¹HNMR (400 MHz, d₆-DMSO+TFA) δ1.27-1.69 (5H, m), 1.90-2.06 (1H, m),2.55-2.87 (2H, m), 3.13-3.21 (2H, m), 4.31-5.26 (6H, m), 7.12-7.48 (6H,m), 7.84-7.86 (2H, m), 8.08-8.58 (1H, m) ; ¹³C NMR (100 MHz, DMSO+TFA)δ20.5 (CH₂), 24.2 (CH₂), 27.73 (CH₂), 34.6/34.8 (CH₂), 44.9 (CH₂),48.5/48.7 (CH), 52.1/52.5 (CH), 55.4/55.7 (CH), 84.2 (d, CH₂F), 120.2(CH), 123.3 (CH), 123.6 (CH), 124.7 (CH), 126.1 (C),126.3 (CH), 128.0(CH), 128.3 (CH), 128.7 (CH), 131.6 (C), 134.6 (C), 140.2 (C),172.1/172.2 (CO), 172.4/172.4 (CO), 203.0 (d, CO) ; ¹⁹F NMR(376 MHz,DMSO+TFA) δ−226.8 (t), −226.9 (t), −232.7 (t), −232.9 (t).

EXAMPLE 8 [3S/R, (2S)]-3-{[1-(2-Methylphenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoic acid

[0102]

[0103] The title compound was prepared from 2-methylphenothiazinecarbonyl chloride using procedures similar to those described above inMethods A-E (3.7 mg, 17%): ¹H NMR (400 MHz, d₆-DMSO) δ0.86-1.75 (7H, m),1.91-2.80 (5H, m), 3.13-3.66 (2H, m), 4.13-4.77 (2H, m), 5.06-5.33 (1H,m), 6.96-8.61(8H, m); ¹³C NMR (100 MHz, d₆-DMSO) δ201.3, 201.2, 201.0,171.8, 170.7, 170.6, 170.2, 169.8, 169.7, 156.2, 155.8, 139.7, 139.6,139.6, 139.5, 136.1, 127.5, 125.9, 122.3, 122.2, 120.6, 119.8, 83.7,83.6, 81.9, 81.8, 66.2, 53.8, 50.5, 43.1, 33.1, 28.6, 26.1, 25.9, 22.5,22.3, 22.2, 22.1, 21.2 and 18.9; ¹⁹F NMR (376 MHz, d₆-DMSO) δ−226.7,−226.8, −230.2, −231.2, −232.7, −233.0.

EXAMPLE 9 [3S/R,(2S)]-3-{[1-(2-Trifluoromethylphenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoic acid

[0104]

[0105] The title compound was prepared from2-trifluoromethylphenothiazine carbonyl chloride using proceduressimilar to those described above in Methods A-E (1.78 g, 98%):IR (solid,cm⁻¹) 1792.9, 1654.6, 1465.2, 1403.7, 1321.8, 1224.5, 1163.0, 1116.9; ¹HNMR (400 MHz, d₆-DMSO) δ0.76-1.09 (1H, m), 1.20-1.69 (4H, m), 1.87-2.10(1H, m), 2.50-2.98 (2H, m), 3.18 (1H, m), 3.41-3.62 (1H, m), 4.26-4.80(2.75H, m), 5.02-5.32 (1.25H, m), 7.17-7.64 (6H, m), 8.03-8.20 (1H, m),8.43-8.68 (1H, brm), 12.50 (1H, brs); ¹⁹F NMR (376 MHz, d₆-DMSO) δ−61.6,−61.64, −61.7, −226.8, −226.9, −230.2, −231.3, −232.8, −233.1.

EXAMPLE 10 [3S/R, (2S)]-3-{[1-(2-Methoxyphenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-4-fluoro-4-oxo-pentanoic acid

[0106]

[0107] The title compound was prepared from 2-methoxyphenothiazinecarbonyl chloride using procedures similar to those described above inMethods A-E (21 mg, 4%):IR (solid, cm⁻¹) 1174.6, 1205.4, 1263.8, 1405.7,1442.81, 1463.7, 1596.9, 1652.8; ¹H NMR (400 MHz, CDCl3) δ1.1 (1H, m),1.3 (1H, m), 1.5-1.7 (3H, m), 2.15 (1H, m), 2.7-3.0 (3H, m), 3.60 (1H,t), 3.85 (3H, s), 4.6-4.9 (4H, m), 6.75 (1H, d), 7.1-7.7 (7H, m) ; ¹³CNMR (100 MHz, CDCl₃) δ20.9, 23.9, 26.0, 46.1, 56.1, 57.1, 109.8, 112.1,112.2, 123.6, 126.9, 126.2, 127.9, 128.3, 128.6, 128.7, 141.1, 141.2,142.4, 159.8, 159.9; ¹⁹F NMR (376 MHz, CDCl₃) δ−231.09 and −230.76.

EXAMPLE 11 [3S/R, (2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentamide

[0108]

[0109] To a stirred mixture of [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoic acid (200 mg, 0.4 mmol), prepared as inExample 1, in anhydrous THF (4m1), was added EDC (84 mg, 0.44 mmol), anda solution of the ammonia in 1,4 dioxane (0.8 ml of a 0.5M solution, 4mmol). The mixture was stirred at room temperature for 16 h thenconcentrated under reduced pressure. The residue was purified by flashchromatography (6% Methanol in dichloromethane) to afford the titlecompound as a white solid (35.5 mg, 18%): ¹H NMR (400 MHZ, CDCl₃)δ0.74-1.69 (6H, m), 2.02-3.63 (4H, m), 5.15-4.72 (4H, m), 6.05-6.79 (4H,4xs) and 7.11-7.79 (8H, m). ¹⁹F (376 MHz, CDCl₃) δ−225.45, −225.60,−227.42, −228.07, −228.09 and −231.65.

EXAMPLE 12 [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid ethyl amide

[0110]

[0111] To a stirred mixture of [3S/R, (2S)]-5-fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid (196 mg, 0.4 mmol), prepared as in Example 1, in anhydrous DCM (8ml), was added Polymer bound EDC (400 mg, 0.8 mmol) and a solution ofthe ethylamine in THF (0.6 ml of a 2M solution, 12 mmol). The mixturewas stirred at room temperature for 16 h then concentrated under reducedpressure. The residue was purified by flash chromatography (2.5%methanol in dichloromethane) to afford the title compound as a whitesolid (22.7 mg, 11%): ¹H NMR (400 MHz, CDCl₃) δ1.09-1.39 (3H, m),1.44-1.66 (6H, m), 2.04-3.66 (7H, m), 4.18-4.76 (6H, m) and 7.10-7.79(8H, m). ¹⁹F NMR (376 MHz, CDC₃) δ−223.32, −223.73, −225.92 and −226.76.

EXAMPLE 13 [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid diethyl amide

[0112]

[0113] To a stirred mixture of [3S/R, (2S)]-5-fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid (200 mg, 0.4 mmol), prepared as in Example 1, in anhydrous DCM (4ml), was added carbodiimide (74 mg, 0.44 mmol) and a diethylamine (0.62ml, 6 mmol). The mixture was stirred at room temperature for 16 h thenconcentrated under reduced pressure. The residue was purified by flashchromatography (2.5% Methanol in dichloromethane) to afford the titlecompound as a white solid (25.3 mg, 11%): ¹H NMR (400 MHz, CDCl₃)δ1.00-1.23 (6H, m), 1.45-1.67 (6H, m), 2.18 (1H, m), 2.74 (1H, m), 2.92(1H, m), 3.21-3.33 (4H,m), 3.65 (1H, m), 4.68 (1H, m), 4.85 (1H, m),4.98-5.36 (2H, m) and 7.12-7.95 (8H, m). ¹⁹F NMR (376 MHz, CDCl₃)δ−232.37 and −232.72.

EXAMPLE 14 [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid N,N-dimethyl aminoethylamide.

[0114]

[0115] To a stirred mixture of [3S/R,(2S)]-5-fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoic acid (100 mg, 0.2 mmol), prepared as inExample 1, in anhydrous DCM (5 ml), was added Polymer bound EDC (300 mg,0.6 mmol) and N,N-dimethyl propylamide (0.88 ml, 6 mmol). The mixturewas stirred at room temperature for 16 h then concentrated under reducedpressure. The residue was purified by flash chromatography (2% Methanolin dichloromethane) to afford the title compound as a white solid (16.5mg, 14%): ¹H NMR (400 MHz, CDCl₃) δ1.48-1.68 (6H, m), 2.20-2.40 (8H, m),2.60 (1H, m), 2.71-3.06 (3H, m); 3.73 (1H, m), 4.01 (1H, m), 4.16-4.38(2H, m), 4.62 (1H, m), 4.75 (1H, m), 6,96 (1H, m) and 7.11-7.77 (8H, m).¹⁹F NMR (376 MHz, CDCl₃) δ−222.48 and −222.63.

EXAMPLE 15 [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoicacid N methyl piperazine amide.

[0116]

[0117] To a stirred mixture of [3S/R,(2S)]-5-fluoro-4-oxo-3-{[1-(phenothiazine-10-carbonyl)piperidine-2-carbonyl]amino}-pentanoic acid (200 mg, 0.4 mmol), prepared as inExample 1, in anhydrous THF (4 ml), was added EDC (84 mg, 0.44 mmol) andN methyl piperazine (0.88 ml, 8 mmol). The mixture was stirred at roomtemperature for 16 h then concentrated under reduced pressure. Theresidue was purified by flash chromatography (2% Methanol indichloromethane) to afford the title compound as a white solid (33 mg,14%): ¹H NMR (400 MHz, CDCl₃) δ1.47-1.70 (6H, m), 2.13-2.50 (7H, m),2.75 (1H, m), 2.90 (1H, m), 3.19 (1H, m), 3,41-3.65 (5H, m), 4.66 (1H,m), 4.86 (1H, m), 4.97-5.34 (2H, m), 7.12-7.18 (2H, m), 7.27-7.37 (3H,m), 7.23-7.54 (1H, m) and 7.74-7.78 (2H, m). ¹⁹F NMR (376 MHz, CDCl₃)δ−232.24 and −232.52.

[0118] The compounds of this invention are designed to inhibit caspases.Therefore, the compounds of this invention may be assayed for theirability to inhibit apoptosis, the release of IL-β or caspase activitydirectly. Assays for each of the activities are known in the art and aredescribed below in detail in the Testing section.

[0119] According to another embodiment, the invention provides acomposition comprising a compound of this invention or apharmaceutically acceptable salt thereof, as described above, and apharmaceutically acceptable carrier.

[0120] If pharmaceutically acceptable salts of the compounds of thisinvention are utilized in these compositions, those salts are preferablyderived from inorganic or organic acids and bases. Included among suchacid salts are the following: acetate, adipate, alginate, aspartate,benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate,camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth.

[0121] Also, the basic nitrogen-containing groups may be quaternizedwith such agents as lower alkyl halides, such as methyl, ethyl, propyl,and butyl chloride, bromides and iodides; dialkyl sulfates, such asdimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides suchas decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides,aralkyl halides, such as benzyl and phenethyl bromides and others. Wateror oil-soluble or dispersible products are thereby obtained.

[0122] The compounds utilized in the compositions and methods of thisinvention may also be modified by appending appropriate functionalitiesto enhance selective biological properties. Such modifications are knownin the art and include those which increase biological penetration intoa given biological system (e.g., blood, lymphatic system, centralnervous system), increase oral availability, increase solubility toallow administration by injection, alter metabolism and alter rate ofexcretion.

[0123] Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

[0124] According to a preferred embodiment, the compositions of thisinvention are formulated for pharmaceutical administration to a mammal,preferably a human being.

[0125] Such pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally or intravenously.

[0126] Sterile injectable forms of the compositions of this inventionmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

[0127] The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers that are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

[0128] Alternatively, the pharmaceutical compositions of this inventionmay be administered in the form of suppositories for rectaladministration. These may be prepared by mixing the agent with asuitable non-irritating excipient which is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

[0129] The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

[0130] Topical application for the lower intestinal tract may beeffected in a rectal suppository formulation (see above) or in asuitable enema formulation. Topically-transdermal patches may also beused.

[0131] For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions may be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

[0132] For ophthalmic use, the pharmaceutical compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with our without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticalcompositions may be formulated in an ointment such as petrolatum.

[0133] The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

[0134] The above-described compositions are particularly useful intherapeutic applications relating to an IL-1 mediated disease, anapoptosis mediated disease, an inflammatory disease, an autoimmunedisease, a destructive bone disorder, a proliferative disorder, aninfectious disease, a degenerative disease, a disease associated withcell death, an excess dietary alcohol intake disease, a viral mediateddisease, uveitis, inflammatory peritonitis, osteoarthritis,pancreatitis, asthma, adult respiratory distress syndrome,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Grave's disease, autoimmune gastritis,diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, chronic active hepatitis, myasthenia gravis,inflammatory bowel disease, Crohn's disease, psoriasis, atopicdermatitis, scarring, graft vs host disease, organ transplant rejection,osteoporosis, leukemias and related disorders, myelodysplastic syndrome,multiple myeloma-related bone disorder, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma,multiple myeloma, haemorrhagic shock, sepsis, septic shock, burns,Shigellosis, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Kennedy's disease, prion disease, cerebral ischemia,epilepsy,myocardial ischemia, acute and chronic heart disease, myocardialinfarction, congestive heart failure, atherosclerosis, coronary arterybypass graft, spinal muscular atrophy, amyotrophic lateral sclerosis,multiple sclerosis, HIV-related encephalitis, aging, alopecia,neurological damage due to stroke, ulcerative colitis, traumatic braininjury, spinal cord injury, hepatitis-B, hepatitis-C, hepatitis-G,yellow fever, dengue fever, or Japanese encephalitis, various forms ofliver disease, renal disease, polyaptic kidney disease, H.pylori-associated gastric and duodenal ulcer disease, HIV infection,tuberculosis, and meningitis. The compounds and compositions are alsouseful in treating complications associated with coronary artery bypassgrafts and as a component of immunotherapy for the treatment of variousforms of cancer.

[0135] The amount of compound present in the above-describedcompositions should be sufficient to cause a detectable decrease in theseverity of the disease or in caspase activity and/or cell apoptosis, asmeasured by any of the assays described in the examples.

[0136] The compounds of this invention are also useful in methods forpreserving cells, such as may be needed for an organ transplant or forpreserving blood products. Similar uses for caspase inhibitors have beenreported (Schierle et al., Nature Medicine, 1999, 5, 97). The methodinvolves treating the cells or tissue to be preserved with a solutioncomprising the caspase inhibitor. The amount of caspase inhibitor neededwill depend on the effectiveness of the inhibitor for the given celltype and the length of time required to preserve the cells fromapoptotic cell death.

[0137] According to another embodiment, the compositions of thisinvention may further comprise another therapeutic agent. Such agentsinclude, but are not limited to, thrombolytic agents such as tissueplasminogen activator and streptokinase. When a second agent is used,the second agent may be administered either as a separate dosage form oras part of a single dosage form with the compounds or compositions ofthis invention.

[0138] It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of active ingredients will also depend upon the particularcompound and other therapeutic agent, if present, in the composition. Asused herein, the term “patient” refers to a warm-blooded animal, such asfor example, rats, mice, dogs, cats, guinea pigs, and primates such ashumans.

[0139] In a preferred embodiment, the invention provides a method oftreating a mammal, having one of the aforementioned diseases, comprisingthe step of administering to said mammal a pharmaceutically acceptablecomposition described above. In this embodiment, if the patient is alsoadministered another therapeutic agent or caspase inhibitor, it may bedelivered together with the compound of this invention in a singledosage form, or, as a separate dosage form. When administered as aseparate dosage form, the other caspase inhibitor or agent may beadministered prior to, at the same time as, or following administrationof a pharmaceutically acceptable composition comprising a compound ofthis invention.

[0140] In order that this invention be more fully understood, thefollowing biological testing examples are set forth. These examples arefor the purpose of illustration only and are not to be construed aslimiting the scope of the invention in any way.

Biolgical Testing Examples EXAMPLE 16 Enzyme Assays

[0141] The assays for caspase inhibition are based on the cleavage of afluorogenic substrate by recombinant, purified human Caspases -1,-3, or-8. The assays are run in essentially the same way as those reported byGarcia-Calvo et al. (J. Biol. Chem. 273 (1998), 32608-32613), using asubstrate specific for each enzyme. The substrate for Caspase-1 isAcetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin. The substrate forCaspases -3, and -8 is Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin.

[0142] The observed rate of enzyme inactivation at a particularinhibitor concentration, k_(obs), is computed by direct fits of the datato the equation derived by Thornberry et al. (Biochemistry 33 (1994),3943-3939) using a nonlinear least-squares analysis computer program(PRISM 2.0; GraphPad software). To obtain the second order rateconstant, kinact, kobs values are plotted against their respectiveinhibitor concentrations and k_(inact) values are subsequentlycalculated by computerized linear regression.

[0143] The compounds tested under Example 16 possess k_(inact) >50000M⁻¹s⁻¹ against caspase-1, caspase-3 and caspase-8.

EXAMPLE 17 Inhibition of IL-1β secretion from Mixed Population ofPeripheral Blood Mononuclear Cells (PBMC)

[0144] Processing of pre-IL-1β by caspase-1 may be measured in cellculture using a variety of cell sources. Human PBMC obtained fromhealthy donors provides a mixed population of lymphocyte and mononuclearcells that produce a spectrum of interleukins and cytokines in responseto many classes of physiological stimulators.

[0145] Experimental Procedure

[0146] The test compound is dissolved in Dimethyl Sulphoxide (DMSO,Sigma#D-2650) to give a 100 mM stock solution. This is diluted in completemedium consisting of RPMI containing 10% heat inactivated FCS (Gibco BRL#10099-141), 2 mM L-Glutamine (Sigma, #G-7513), 100 U penicillin and 100μg/ml streptomycin (Sigma #P-7539). The final concentration range oftest compound is from 100 μM down to 6 nM over eight dilution steps. Thehighest concentration of test compound is equivalent to 0.1% DMSO in theassay.

[0147] Human PBMC are isolated from Buffy Coats obtained from the bloodbank using centrifugation on Ficoll-Paque leukocyte separation medium(Amersham, #17-1440-02) and the cellular assay is performed in a sterile96 well flat-bottomed plate (Nunc). Each well contains 100 μl of thecell suspension, 1×10⁵ cells, 50 μl of compound dilutions and 50 μl ofLPS (Sigma #L-3012) at 50 ng/ml final concentration. Controls consist ofcells +/−LPS stimulation and a serial dilution of DMSO diluted in thesame way as compound. The plates are incubated for 16-18 h at 37° C. in5% CO₂ & 95% humidity atmosphere.

[0148] After 16-18 h the supernatants are harvested after centrifugingthe plates at 100×g at 18° C. for 15 min and assayed for their IL-1μcontent. Measurement of mature IL-1μ in the supernatant is performedusing the Quantikine kits (R&D Systems) according to manufacturer'sinstructions. Mature IL-1β levels of about 600-1500 pg/ml are observedfor PBMCs in positive control wells.

[0149] The inhibitory potency of the compounds may be represented by anIC₅₀ value, which is the concentration of inhibitor at which 50% of themature IL-1β is detected in the supernatant as compared to the positivecontrols.

[0150] Selected compounds from Table 1 were tested according to Example17 and found to provide an IC₅₀ value less than 2 μM for inhibition ofIL-1β secretion from PBMC.

EXAMPLE 18 Anti-Fas Induced Apoptosis Assay

[0151] Cellular apoptosis may be induced by the binding of Fas ligand(FasL) to its receptor, CD95 (Fas). CD95 is one of a family of relatedreceptors, known as death receptors, which can trigger apoptosis incells via activation of the caspase enzyme cascade. The process isinitiated by the binding of the adapter molecule FADD/MORT-1 to thecytoplasmic domain of the CD-95 receptor-ligand complex. Caspase-8 thenbinds FADD and becomes activated, initiating a cascade of events thatinvolve the activation of downstream caspases and subsequent cellularapoptosis. Apoptosis can also be induced in cells expressing CD95 eg theJurkat E6.1 T cell lymphoma cell line, using an antibody, rather thanFasL, to crosslink the cell surface CD95. Anti-Fas-induced apoptosis isalso triggered via the activation of caspase-8. This provides the basisof a cell-based assay to screen compounds for inhibition of thecaspase-8-mediated apoptotic pathway.

[0152] Experimental Procedure

[0153] Jurkat E6.1 cells are cultured in complete medium consisting ofRPMI-1640 (Sigma No) +10% foetal calf serum (Gibco BRL No.10099-141)+2mM L-glutamine (Sigma No. G-7513). The cells are harvested in logphase of growth. 100 ml Cells at 5-8×10⁵ cells/ml are transferred tosterile 50 ml Falcon centrifuge tubes and centrifuged for 5 minutes at100 xg at room temperature. The supernatant is removed and the combinedcell pellets resuspended in 25 ml of complete medium. The cells arecounted and the density adjusted to 2×10⁶cells/ml with complete medium.

[0154] The test compound is dissolved in dimethyl sulphoxide(DMSO)(Sigma No. D-2650) to give a 100 mM stock solution. This isdiluted to 400 μM in complete medium, then serially diluted in a 96-wellplate prior to addition to the cell assay plate.

[0155] 100 μl of the cell suspension (2×10⁶ cells) is added to each wellof a sterile 96-well round-bottomed cluster plate (Costar No. 3790). 50μl of compound solution at the appropriate dilution and 50 μl ofanti-Fas antibody, clone CH-11 (Kamiya No.MC-060) at a finalconcentration of 10 ng/ml, are added to the wells. Control wells are setup minus antibody and minus compound but with a serial dilution of DMSOas vehicle control. The plates are incubated for 16-18 hrs at 37° C. in5% CO₂ and 95% humidity.

[0156] Apoptosis of the cells is measured by the quantitation of DNAfragmentation using a ‘Cell Death Detection Assay’ fromBoehringer-Mannheim, No. 1544 675. After incubation for 16-18 hrs theassay plates are centrifuged at 100×g at room temperature for 5 minutes.150 μl of the supernatant are removed and replaced by 150 μl of freshcomplete medium. The cells are then harvested and 200 μl of the lysisbuffer supplied in the assay kit are added to each well. The cells aretriturated to ensure complete lysis and incubated for 30 minutes at 4°C. The plates are then centrifuged at 1900×g for 10 minutes and thesupernatants diluted 1:20 in the incubation buffer provided. 100 μl ofthis solution is then assayed exactly according to the manufacturer'sinstructions supplied with the kit. OD₄₀₅nm is measured 20 minutes afteraddition of the final substrate in a SPECTRAmax Plus plate reader(Molecular Devices). OD₄₀₅nm is plotted versus compound concentrationand the IC₅₀ values for the compounds are calculated using thecurve-fitting program SOFTmax Pro (Molecular Devices) using the fourparameter fit option. Selected compounds from Table 1 were testedaccording to Example 18 and found to provide an IC₅₀ value less than 100nM for the activity in the FAS induced apoptosis assay.

[0157] While we have described a number of embodiments of thisinvention, it is apparent that our basic examples may be altered toprovide other embodiments, which utilize the compounds and methods ofthis invention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments, which have been represented by way of example.

What is claimed is:
 1. A compound of formula I:

wherein: Ring A is an optionally substituted piperidine,tetrahydroquinoline or tetrahydroisoquinoline ring; R¹ is hydrogen, CN,CHN₂, R, or CH₂Y; R is an optionally substituted group selected from analiphatic group, an aryl group, or an aralkyl group; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; and R³ is hydrogen, an optionally substituted arylgroup, an optionally substituted aralkyl group or an optionallysubstituted C₁₋₆ aliphatic group, R⁴ is an optionally substituted groupselected from an aryl group or a heterocyclyl group, or R³ and R⁴ takentogether with the nitrogen to which they are attached optionally form asubstituted or unsubstituted monocyclic, bicyclic or tricyclic ring. 2.The compound according to claim 1 having one or more features selectedfrom the group consisting of: (a) R¹ is CH₂Y where Y is anelectronegative leaving group; (b) R² is CO₂H, esters, amides orisosteres thereof; and (c) R³ is a hydrogen atom, an optionallysubstituted aryl group, an optionally substituted aralkyl group or anoptionally substituted C₁₋₆ aliphatic group, R⁴ is an optionallysubstituted group selected from an aryl group or a heterocyclyl group,or R³ and R⁴, taken together with the nitrogen to which they areattached, optionally form a ring selected from the group consisting ofindole, isoindole, indoline, indazole, purine, benzimidazole,benzthiazole, imidazole, imidazoline, thiazole, pyrrole, pyrrolidine,pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole, piperidine,morpholine, thiomorpholine, piperazine, carbazole, phenothiazine,phenoxazine, phenanthridine, acridine, purine, quinolizine, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,1,8-naphthyridine, pteridine, quinuclidine, and phenazine.
 3. Thecompound of claim 2, wherein: (a) R¹ is CH₂Y where Y is anelectronegative leaving group; (b) R² is CO₂H, esters, amides orisosteres thereof; and (c) R³ is a hydrogen atom, an optionallysubstituted aryl group, an optionally substituted aralkyl group or anoptionally substituted C₁₋₆ aliphatic group, R⁴ is an optionallysubstituted group selected from an aryl group, or a heterocyclyl group;or R³ and R⁴, taken together with the nitrogen to which they areattached, optionally form a ring selected from the group consisting ofindole, isoindole, indoline, indazole, purine, benzimidazole,benzthiazole, imidazole, imidazoline, thiazole, pyrrole, pyrrolidine,pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole, piperidine,morpholine, thiomorpholine, piperazine, carbazole, phenothiazine,phenoxazine, phenanthridine, acridine, purine, quinolizine, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,1,8-naphthyridine, pteridine, quinuclidine, and phenazine.
 4. Thecompound according to claim 3 wherein —CH₂Y is —CH₂F.
 5. The compoundaccording to claim 4 wherein R³ and R⁴, taken together with the nitrogento which they are attached, form a ring selected from the groupconsisting of indole, isoindole, indoline, indazole, purine,benzimidazole, benzthiazole, imidazole, imidazoline, thiazole, pyrrole,pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole,piperidine, morpholine, thiomorpholine, piperazine, carbazole,phenothiazine, phenoxazine, phenanthridine, acridine, purine,quinolizine, quinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, quinuclidine,and phenazine.
 6. A composition comprising a compound according to anyof claims 1-5 and a pharmaceutically acceptable carrier.
 7. A method fortreating a disease in a patient that is alleviated by treatment with acaspase inhibitor, which method comprises administering to a patient inneed of such a treatment a therapeutically effective amount of acompound of formula I:

wherein: Ring A is an optionally substituted piperidine,tetrahydroquinoline or tetrahydroisoquinoline ring; R¹ is hydrogen, CN,CHN₂, R, or CH₂Y; R is an optionally substituted group selected from analiphatic group, an aryl group or an aralkyl group; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; and R³ is hydrogen, an optionally substituted arylgroup, an optionally substituted aralkyl group, or an optionallysubstituted C₁₋₆ aliphatic group, R⁴ is an optionally substituted groupselected from an aryl group or a heterocyclyl group, or R³ and R⁴ takentogether with the nitrogen to which they are attached optionally form asubstituted or unsubstituted monocyclic, bicyclic or tricyclic ring. 8.The method according to claim 7 wherein the compound has one or more ofthe following features: (a) Ring A is an optionally substitutedpiperidine; (b) R¹ is CH₂Y where Y is an electronegative leaving group;(c) R² is CO₂H, esters, amides or isosteres thereof; and (d) R³ is ahydrogen atom, an optionally substituted aryl group, an optionallysubstituted aralkyl group or an optionally substituted C₁₋₆ aliphaticgroup; R⁴ is an optionally substituted group selected from an arylgroup, a heterocyclyl group; or R³ and R⁴, taken together with thenitrogen to which they are attached, optionally form a ring selectedfrom the group consisting of indole, isoindole, indoline, indazole,purine, benzimidazole, benzthiazole, imidazole, imidazoline, thiazole,pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine,triazole, piperidine, morpholine, thiomorpholine, piperazine, carbazole,phenothiazine, phenoxazine, phenanthridine, acridine, purine,quinolizine, quinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, quinuclidine,and phenazine.
 9. The method according to claim 8 wherein: (a) Ring A isan optionally substituted piperidine; (b) R¹ is CH₂Y where Y is anelectronegative leaving group; (c) R² is CO₂H, esters, amides orisosteres thereof; and (d) R³ is a hydrogen atom, an optionallysubstituted aryl group, an optionally substituted aralkyl group or anoptionally substituted C₁₋₆ aliphatic group, R⁴ is an optionallysubstituted group selected from an aryl group or a heterocyclyl group,or R³ and R⁴, taken together with the nitrogen to which they areattached, optionally form a ring selected from the group consisting ofindole, isoindole, indoline, indazole, purine, benzimidazole,benzthiazole, imidazole, imidazoline, thiazole, pyrrole, pyrrolidine,pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole, piperidine,morpholine, thiomorpholine, piperazine, carbazole, phenothiazine,phenoxazine, phenanthridine, acridine, purine, quinolizine, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,1,8-naphthyridine, pteridine, quinuclidine, and phenazine.
 10. Themethod according to any of claims 7-9 wherein the compound is used totreat a disease selected from an IL-1 mediated disease, an apoptosismediated disease, an inflammatory disease, an autoimmune disease, adestructive bone disorder, a proliferative disorder, an infectiousdisease, a degenerative disease, a disease associated with cell death,an excess dietary alcohol intake disease, a viral mediated disease,uveitis, inflammatory peritonitis, osteoarthritis, pancreatitis, asthma,adult respiratory distress syndrome, glomerulonephritis, rheumatoidarthritis, systemic lupus erythematosus, scleroderma, chronicthyroiditis, Grave's disease, autoimmune gastritis, diabetes, autoimmunehemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronicactive hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn'sdisease, psoriasis, atopic dermatitis, scarring, graft vs host disease,organ transplant rejection, osteoporosis, leukemias and relateddisorders, myelodysplastic syndrome, multiple myeloma-related bonedisorder, acute myelogenous leukemia, chronic myelogenous leukemia,metastatic melanoma, Kaposi's sarcoma, multiple myeloma, haemorrhagicshock, sepsis, septic shock, burns, Shigellosis, Alzheimer's disease,Parkinson's disease, Huntington's disease, Kennedy's disease, priondisease, cerebral ischemia, epilepsy, myocardial ischemia, acute andchronic heart disease, myocardial infarction, congestive heart failure,atherosclerosis, coronary artery bypass graft, spinal muscular atrophy,amyotrophic lateral sclerosis, multiple sclerosis, HIV-relatedencephalitis, aging, alopecia, neurological damage due to stroke,ulcerative colitis, traumatic brain injury, spinal cord injury,hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, orJapanese encephalitis, various forms of liver disease, renal disease,polyaptic kidney disease, H. pylori-associated gastric and duodenalulcer disease, HIV infection, tuberculosis, meningitis, a treatment forcomplications associated with coronary artery bypass grafts, or animmunotherapy for the treatment of various forms of cancer.
 11. Themethod according to any of claims 7-9 wherein the compound is used totreat complications associated with coronary artery bypass grafts. 12.The method according to any of claims 7-9 wherein the compound is usedfor the preservation of cells, said method comprising the step ofbathing the cells in a solution of the compound or a pharmaceuticallyacceptable derivative thereof.
 13. The method according to any of claims7-9 wherein the compound is used for an organ transplant or forpreserving blood products.
 14. The method according to any of claims 7-9wherein the compound is used as a component of immunotherapy for thetreatment of cancer.